Project Summary Capsaicin- and heat-activated TRPV1 ion channel is a primary nociceptor for both chemical and thermal stimuli, hence an attractive target for pain medication. Despite the availability of cryo-EM structures of TRPV1 at up-to-2.9 resolutions, molecular mechanisms underlying TRPV1 activation remains unclear. A major limitation for obtaining mechanistic information from the cryo-EM structures is the general lack of resolution to determine side-chain orientation and the associated atomic interaction. We recently demonstrated that the limitation could be overcome by combining Rosetta structural prediction with site-specific functional tests such as thermodynamic mutant cycle analysis that serve to constrain, validate, and improve structure prediction. Using this iterative approach, in combination with molecular dynamics (MD) simulation and site-specific fluorescence recordings including FRET and patch fluorometry, our proposed study aims to identify functional interactions in the capsaicin-binding domain and the outer pore and, more importantly, to reveal dynamic changes of these interactions during capsaicin- and heat-induced activation. We will take particular advantage of our newly designed fluorescent capsaicin analogs that allow us to directly monitor ligand binding, as well as the fluorescent unnatural amino acid (FUAA) incorporation method to introduce a small fluorophore to the channel's moving parts. Our goal is to elucidate key molecular interactions that mediate chemical and thermal activation, thus providing a molecular framework to guide pharmaceutical intervention.